PROJECT SUMMARY/ABSTRACT Keratoconus is the degeneration of the corneal stroma, which is the layer that provides the structure for the cornea. This degeneration causes the cornea to slowly change from its normal curved shape to a more conical shape, leading to vision distortion. Unfortunately, to-date, the cause of the disease is not known, and in most cases, patients require some kind of surgical intervention. Currently, there are no in vitro or animal models available, limiting the number of feasible studies. Therefore, we propose to develop a novel in vitro 3- dimensional (3D) culture system consisting of human keratoconus cells (HKCs), which will allow us to identify vital proteins and metabolites that are driving the disease in vitro and compare these results to in vivo tear samples. Our preliminary data consists of some exciting observations that potentially link the defects in vivo to in vitro findings. Firstly, we have identified significant inherent cellular defects in HKCs when compared to normal primary human corneal fibroblasts (HCFs). HKCs were unable to secrete and self-assemble a functional extracellular matrix (ECM) when stimulated with vitamin C (VitC), while HCFs assembled high amounts of collagenous ECM. Secondly, we identified TGF-3 (T3) as a potential growth factor for stimulating HKCs to self-assemble an ECM. Thirdly, we have identified metabolites and proteins that are similarly regulated in our in vitro model when compared to in vivo, some of which may be new markers for identifying keratoconus disease. The hypotheses we propose to test are as follows: (1) T3 stimulates HKCs to adopt a keratocyte-like phenotype and secrete a functional ECM, (2) Regulation of metabolic activity is a key component in the understanding of Keratoconus defects and (3) Identification of new markers for keratoconus disease is vital for early detection and treatment. We propose three specific aims in order to address the following questions: First, what are the inherent differences of HKCs and how may these be regulated by T3? Second, can metabolic activity regulate HKCs to assemble ECM? Third, what are the key proteins regulating keratoconus in vivo and in vitro? The novelty of this proposal is that for the first time, we can establish a 3D in vitro model that can be used to represent the events during keratoconus disease in vivo. Indirect- immunofluorescence microscopy, real-time PCR and transmission electron microscopy will be the major analysis tools for the in vitro model. Metabolomics and Proteomics will be the tools for analyzing both the in vitro and in vivo samples. The combination of the above will link the in vitro to in vivo studies and provide new, novel markers for the diagnosis and prevention of keratoconus at early stages. Relevance to Public Health - Improvement in the ability to detect and diagnose suspicious to advanced keratoconus will enable the exclusion of patients at risk for corneal ectasia after corneal refractive surgery. Also, earlier detection of forme fruste keratoconus may lead to earlier intervention.